This proposal seeks to evaluate the role of KiSS-1 in the age-related changes in responsiveness of GnRH neurons to estradiol (E2) in the female rat. Although strong evidence indicates that KiSS-1 and GPR54 are critical for E2 feedback onto GnRH neurons in young adults across many species including humans, we have virtually no understanding of how this mechanism may change during the middle-age transition towards reproductive senescence. The following specific aims will address the hypothesis that a decline in responsiveness of KiSS-1 neurons to E2 plays a critical role in mediating the reduced magnitude and delay of the LH surge in aging female rats. Our first aim will be to determine the effect of age and time of day on KiSS-1. We will investigate whether the production of KiSS-1 and estrogen receptors (ERs) from KiSS-1 neurons, or KiSS-1 and ER cell density change with age in the AVPV. We will determine whether there is a diurnal variation in KiSS-1 gene and protein expression. Additionally we will examine the effect of age and time of day on KiSS-1 axonal density to GnRH neuronal populations critical for the LH surge. Our second aim will be to evaluate the KiSS-1 neuron response to E2 in the AVPV with age. We will accomplish this aim by testing the capacity of E2 to activate KiSS-1 neurons in the AVPV with age and by determining to what extent E2-induced KiSS-1 neuronal activation depends on time of day. Additionally, we will determine whether aging, E2, and time of day differentially affect GPR54 expression in the GnRH neuronal populations of the organum vasculosum of the lamina terminalis/rostral medial preoptic nucleus (OVLT/rMPN), a region known to be involved in generating the GnRH/LH surge. Our third, and final aim, will be to evaluate the effect of age and time of day on the capacity of KiSS-1 to mediate an E2-induced LH surge. In this aim it will be critical to assess whether KiSS-1, given centrally, will elicit a GnRH/LH surge in middle-aged rats and if time of day is a critical factor. Furthermore, we will determine whether age and time of day differentially affect the capacity of centrally administered KiSS-1 to activate GnRH neurons. It is possible that GnRH neurons of middle-aged animals will be less responsive to KiSS-1, so we will examine if GPR54 expression changes with age and time of day. Findings from the proposed study will have critical implications to the understanding of the neuroendocrine involvement of the onset of menopause and age-related infertility in women.